PF-382

PF-382 Cell Line With Phenotypic and Functional Features of Suppressor Lymphocytes

The PF-382 cells grew in single-cell suspension; their doubling time was estimated at 40 hours and did not change appreciably throughout 10 months of continuous culture. On Giemsa-stained preparations the cells appeared homogeneous with little variation in size; they were oval and irregularly shaped and had a high nuclear-to-cytoplasmic ratio. The cytoplasm was basophilic without granules, and the nucleus showed a fine chromatin design and 1 or 2 nucleoli. PF-382 cells were negative for myeloperoxidase, ANAE, and PAS stains and positive for acid phosphatase stains.

This analysis was performed on a total of 34 G-banded metaphases of PF-382 cells that had been in continuous culture for 6 months, Thirty metaphases showed a 46X, Xq-,15p+ karyotype (Fig. 1); two were 45X and two were 47X, Xq-,15p+,+16. Chromosome analysis performed on fresh cells after 10 days in culture showed the same predominant karyotype. On C-banding the normal #15 and the 15p+ were equally stained in the centromeric region, whereas the additional chromatin of the abnormal #15 was not darkly stained.

The only difference with the primary cells was the reduction or almost total disappearance of the OKT4 and OKTll antigens and the E-rosette-forming capability. Thus the cell line shows an early intrathymic phenotype (OKT6+, Leu-l +, Leu9+, OKT3-, OKTll-) with the concomitant expression of the OKT8 antigen. The cells do not express an NK phenotype as indicated by a low or absent reactivity with the Leu-7, B73.1, and OKMI MoAb.

Fig. 1 Representative metaphases of PF-382 cells. (Pegoraro L, et al.,1985)

Activation of the LMO2 Oncogene in T-Cell ALL

LMO2 is a prominent oncogene in T-cell ALL (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples.

LMO2 expression was assessed by qRT-PCR in several T-ALL cell lines arrested at different stages of thymic differentiation. The early T-cell progenitor (ETP)-like T-ALL cell line Loucy expressed LMO2 at levels significantly higher than the more mature T-ALL cell lines (DND-41, ALL-SIL, Jurkat), reflecting physiological expression of LMO2 at the ETP stage of thymic development (Fig. 2A). The TAL1-positive cell lines DU.528 and PF-382 both exhibited upregulated LMO2 expression, yet crucially have no reported chromosomal lesions affecting this locus (Fig. 2A). In contrast to Loucy cells, aberrant H3K27ac marks (indicative of active chromatin) were identified before and encompassing the noncoding exon 2 of the LMO2 gene by ChIP-seq in PF-382 and DU.528 T-ALL cell lines (Fig. 2B). Sequencing across these peaks revealed a heterozygous 20 bp duplication in PF-382 cells and a heterozygous 1 bp deletion in DU.528 cells located close to a region recently described as an intermediate promoter for reasons that were not then apparent (Fig. 2B).

To assess whether the mutations form aberrant sites of MYB binding, ChIP-seq for MYB was performed, and analyzed peaks of MYB enrichment at the LMO2 locus. There was a complete absence of MYB binding at the intermediate promoter in cells that were wild-type at this locus, suggesting that the presence of the single native MYB motif in itself is insufficient to recruit MYB. In contrast, both PF-382 and DU.528 cells that harbor dual MYB motifs displayed precisely aligned MYB binding at the mutation site (Fig. 2B). To determine whether the mutations affected promoter usage, rapid amplification of 5' complementary DNA ends in LMO2 mutant and wild-type cell lines were performed by using a common primer in exon 6 capable of capturing the transcription start site of all LMO2 isoforms. Whereas the majority (73%) of 5' capped transcripts in Loucy cells originated from the proximal promoter, both PF-382 and DU.528 cells demonstrated preferential usage of the recently described intermediate promoter (75% and 67% of transcripts, respectively; Fig. 2C).

Fig. 2 LMO2 intron 1 mutation in T-ALL. (Bento L, et al., 2022)